REPORT by Branimir - 30.09.2004
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1. As we have previously agreed, Ral complexes with Ras were used as the starting point. Pdb structure of RalE41K-Ras complex was extracted from Protein Structural Databse (code 1LFD). Since there are two complexes in unit cell, one of the them has been deleted and Lys 31 in Ral has been mutated back to Glu. Water molecules included in the calculations were selected using InsightII (as Ting suggested).
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2. All the mutants of Ral-Ras complexes have been made according to Table 3 in Serrano's paper. Since all the mutation are to Ala (in two cases there are mutations from Tyr to Phe, but that's even easier), I have made them by editing pdb file. It is important to sterss that in Serrano's paper they have shifted all the residue numbers by 4 (for example His 49 in their table is acctually His 53 in the pdb file).
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3. GNP parameters were obtained using Antechamber. They were very similar to those that Ting obtained for GTP, but different to those that Heather Carlson used for ATP. GNP addopted very strange conformation during the minimization with those parameters, so I finally edit them manually to be more similar to those that H. Carlson used for ATP. For Mg ion I used larger radius from CHARMM, because othervise Mg ion moves to much from the position that has in the crystal structure. With new GNP and Mg parameters I have finally managed to obtained resonable structures in which Mg ion is about 2 A from oxygen atoms of GNP, what is similar to the values that I found for about 15 other Ral-GNP structures from PDB.
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4. For all the mutants polar hydrogens were added by WHATIF. In all complexes the same flips of about seven Asn have been made, and in the most of the complexes His 53 of Ras is double protonated (exceptions are mutants ralN23A, ralN25A, ralK28A in which His53 is monoprotonated, and in ralH49A is mutated). WHATIF did not make rotations of the two residues that in Serrano's paper are made by hand (swich of Ser33 OH group from C=O to NH group of Glu237 backbone, and movement of Lys32 from Lys 52 to Tyr 240 and Asp 238), but it should be stressed that they didn't include water molecules, and with water molecules those flips do not seem to be necessary and so crucial. Anyway, Steffan helped me to find structural factors for similar structures (1K8R nad 1UAD) and I will ask Mery or Zoran to help me check the positions of Ser33, Lys32 and His53.
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5. Rest of the hydrogens were added using xleap/tleap and minimization was performed by Sander. In the first run 500 cycles were performed (first 100 cycles of SD then swiched to CG) and all the heavy atoms of proteins and Mg were constrained with force constant of 32 kcal/molA2. In the second run the same procedure was used, only this time nothing was constrained. (input files for minimizations are the same as used in COMBINE tutorial that is available at http://atlas.villa-bosch.de/mcm/projects/uppsala )
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6. After minimization, Anal was used to produce file that containes matrix with interaction energies beetwen each residue (it is important to stress that for Anal Amber7 was used because Amber8 has a bug) for each complex. For those calculations water molecules were excluded from minimized complex structures. GNP was not divided into three groups (sugar, phosfate and base) because that is not an easy task (it is difficult to obtaine top and crd fiels from xleap for such structures) and Ting told me that Sanja should now how to do that. Van der Waals and eletrostatic inermolecular interactions were extracted to file which is used as input file for GOLPE.
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7. Two COMBINE models were obtained, but they are very bad (negative q2 values). I should check everything, try to find a mistake and get better models. Also desolvatation energy was not included into those models.
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8. Using UHBD, eletrostatic potential was calculated for each complex (although that was not neccessary). Further, Ting gave me scripts for performing UHBD calculations in order to obtaine desolvatation energy. First all minimized structures of complexes have to be supeimposed to the wild type (using InsightII) and than top and crd files should be produced using xleap/tleap. From those files, using Amber, qcd files for UHBD should be produced and desolvatation energies calculated.
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